Heat-labile phosphatase

ABSTRACT

The present invention relates to the preparation of a novel heat-labile phosphatase enzyme from the filamentous fungus Aspergillus niger. This A. Niger phosphatase enzyme has a native molecular weight of approximately 80,000 daltons, and is shown by polyacrylamide gel electrophoresis under denaturing conditions to be an alpha-2 dimer consisting of identical subunits of molecular weight of approximately 37,000 daltons each. The native intact enzyme molecule has an isoelectric point (pI) of 4.6, and exhibits optimal functional activity under reaction conditions of neutral to slightly alkaline pH conditions (about pH 7.0 to about pH 8.5). This enzyme has two characteristics which make it valuable in molecular biology laboratory protocols. First, the enzyme is readily inactivated by mild heating conditions (50° C.); and second, the enzyme is highly specific for DNA as a substrate for the hydrolysis reaction; it does not hydrolyze adenosine triphosphate (ATP). This unique characteristic permits the simultaneous dephosphorylation and labeled rephosphorylation of DNA in the presence of polynucleotide kinase and labeled ATP, and eliminates the requirement for a multiplicity of steps in this DNA end-labeling process.

BACKGROUND OF THE INVENTION

The present invention relates to a new phosphatase enzyme. Inparticular, the present invention relates to the discovery of a novelheat-labile phosphatase enzyme from the filamentous fungus Aspergillusniger. This new phosphatase enzyme is used as a highly-specific reagentfor the hydrolytic removal of terminal phosphate groups from linear DNAmolecules during one-step radioactive end-labeling procedures, as wellas during preparation of linear DNA molecules for use in molecularcloning assays.

In the following discussion, a number of citations from professionaljournals are included for the convenience of the reader. While thesecitations will more fully describe the state of the art to which thepresent invention pertains, the inclusion of these citations is notintended to be an admission that any of the cited publications representprior art with respect to the present invention.

To place this new microbe-derived phosphatase enzyme in perspective, itwill be helpful to provide some background for the molecular processesin which phosphate groups are transferred among cytoplasmic constituentsin a normal cell. In the action of many polypeptide hormones, acritically-important cellular mediator is the "second messenger" proteincalled cyclic adenosine monophosphate (cAMP), which is formed fromadenosine triphosphate (ATP) by the enzyme adenylate cyclase; thislatter enzyme is bound to the cytoplasmic side of plasma membranes (seeWatson et al., Molecular Biology of the Gene (4th Edition), TheBenjamin/Cummings Publishing Company, Menlo Park, Calif., 1987 . Afterits synthesis, cAMP works by stimulating the activity of cAMP-dependentenzymes called protein kinases. Protein kinases are enzymes thattransfer the high-energy terminal phosphate group of ATP to specificamino acids (serine, threonine, or tyrosine residues) on targetproteins. Phosphorylation (i.e., the process of adding a phosphate groupto a protein) alters the enzymatic activities of these target proteins,and, depending on the particular enzymes involved and the location ofthe added phosphate moiety, can either raise or lower their functionalactivities.

In a like manner, removal of a phosphate group from a protein (a processknown as "dephosphorylation") can also greatly modify the functions andactivities of certain biological molecules. Dephosphorylation occurs bya process of "hydrolysis" in which the phosphate group is catalyticallybroken away ("lysed") from a parent molecule by the enzymatic additionof a water molecule to the parent molecule. This ongoing and cyclicprocess of phosphorylation followed by dephosphorylation, as well asdephosphorylation followed by rephosphorylation, are essential processesin the energy-efficient functioning of all living cells.

The dephosphorylation of a linear DNA molecule by the removal of thehighly-reactive terminal 5'-phosphate group is an essential step in anumber of molecular cloning protocols. Removal of the highly-reactiveterminal phosphate prevents the linear DNA molecule from spontaneouslyligating to the 3'-hydroxyl group at the opposite end of the samemolecule, or to terminal hydroxyl groups on other reactive "bystander"DNA molecules in the same reaction mixture. In addition to thisimportant "house-keeping" function, the dephosphorylation reaction isused by research investigators to prepare the reactive 5'-ends of alinear DNA molecule for subsequent radioactive end-labeling in thepresence of polynucleotide kinase and [gamma-³² P]-ATP, as will bediscussed more fully hereinafter.

Currently, the enzyme most widely used in molecular biology protocolsfor the removal of the terminal 5'-end phosphates from DNA molecules iscalf intestine alkaline phosphatase (as discussed in the recent volumeby Maniatis T, Fritsch EF, and Sambrook J (editors): Molecular Cloning:A Laboratory Manual; New York: Cold Spring Harbor Laboratory, pages133-134, 1982). When used in any of a variety of molecular cloningprocedures, this bovine-derived enzyme has the advantage over otherpreviously-used phosphatases in that it can be completely denatured(with total loss of activity) by heating the reaction mixture to 68° C.in the presence of an additional denaturing agent such as thenegatively-charged detergent sodium dodecyl sulfate (SDS). Under theseconditions, the calf intestine alkaline phosphatase is completelydestroyed without denaturing the DNA in the reaction mixture. This isimportant because the native calf enzyme has the capacity to react witha wide variety of phosphate-bearing substrates, including theenergy-rich molecule adenosine triphosphate (ATP). Because of thiscapacity to react with ATP, it is absolutely necessary to inactivate orremove the calf phosphatase after the dephosphorylation reaction iscomplete, in order to prevent its interfering with phosphate transferfrom ATP in the subsequent reaction steps of an end-labeling protocol.

The fairly low inactivation temperature of 68° C. is an important factoin the current selection of calf intestine alkaline phosphatase for usein molecular cloning and end-labeling protocols. Other alkalinephosphatases, derived from such sources as the bacterium Escherichiacoli, can only be inactivated by boiling the reaction mixture in whichthat enzyme is contained. Such harsh temperatures (at least 100° C.) arelikely to denature more than just the phosphatase enzyme, and may causeirreversible damage to the DNA as well. Furthermore, it is not clearthat boiling is sufficient to completely inactivate the E. coliphosphatase, making the use of this microbial enzyme even lessattractive.

While working with th calf intestine alkaline phosphatase does presentsome significant advantages over using other phosphatase enzymes, thereare disadvantages. For example, the need for the combination of heatingthe reaction mixture to 68° C. and using SDS to inactivate the calfenzyme is cumbersome. In radioactive end-labeling procedures, forexample, use of these procedures necessitates a multiplicity of reactionsteps in order to eliminate the enzyme. The DNA must be precipitatedfrom the reaction mixture, washed, and then re-isolated free of theenzyme before the DNA is used as target substrate for reactivity withother phosphate-bearing molecules in subsequent reaction steps. Thesemultiple steps of heating, precipitating, washing and re-isolating DNAmolecules are a real disadvantage in studies in which such factors astime, or temperature (or both) are critical.

Additional disadvantages which are evident when working with thecalf-derived enzyme, as well as with the E. coli-derived phosphatase,are their broad reaction specificities (i.e., both have the capacity todephosphorylate more than just DNA molecules). Furthermore, it is clearthat enzymes derived from mammalian sources are not as convenient toobtain as they are from microbial sources; accordingly,mammalian-derived enzymes have associated with them certain economicdisadvantages, which, depending on the enzyme source, can be verysignificant. Consequently, there is a need for finding an alternatesource (preferably microbial) for phosphatase enzymes. This has been theobjective of much research.

An important microbial source which has been well studied over the yearsis the filamentous fungus Aspergillus niger (hereinafter "A. niger").Several phosphatase enzymes have been isolated from this microbialorganism, and have been found to exhibit a broad substrate specificity(not unlike that of calf intestine alkaline phosphatase). With regard tofunctional characteristics, they have been generally categorized asbeing either "acid" or "alkaline" phosphatases, i.e., categorizedaccording to the pH value at which their enzymatic function is optimalin the hydrolysis reaction associated with the dephosphorylationprocess: pH 2.5 to 5 and pH 8.5 to 9.5, respectively. Two reports havesuggested the presence of as many as five acid phosphatase activities(i.e., pH 2.5 to 5.0) in extracts of A. niger (Komano T, Plant CellPhysiol. 16: 643-658, 1975; Pathak and Sreenivasan, Arch. Biochem.Biophys. 59: 366-372, 1955); two other reports have indicated thepresence of several alkaline phosphatase activities from A. niger(functional pH optima from pH 8.5 to 10) (Rokosu and Uadia, Int. J.Biochem. 11: 541-544, 1980; Ramaswamy and Bheemeswar, Experientia 32:852-853, 1976). These alkaline phosphatases, like the calf intestinealkaline phosphatase, have also been reported to exhibit a broadhydrolytic reactivity on substrates which include sugar phosphates,nucleotides such as adenosine-5'-phosphate, small synthetic substratessuch as 4-nitrophenylphosphate, and inorganic pyrophosphate (P˜P).

Phosphatase enzymes with a much more limited substrate specificity havealso been found in microorganisms. Examples of such restrictive enzymeactivity are the phosphomonoester hydrolases. One such enzyme is4-nitrophenylphosphatase, which is highly specific for synthetic4-nitrophenylphosphate (4-NPP) as a substrate. While this smallsubstrate molecule is chromogenic before it is hydrolysed by thephosphatase enzyme, its chromophore is revealed only after hydrolysis,and this is shown by a significant increase in the absorption of bluelight by the hydrolysed substrate.

Several distinct forms of this 4-nitrophenylphosphatase enzyme have beenisolated from cells of the yeast Saccharomyces cerevisiae (Attias andBonnet, Biochim. Biophys. Acta 268: 422-430, 1972), and have been foundto function best in an environment having a pH optima between pH 8.0 and8.5. Further characterization of these yeast-derived enzymes has shownthat their functional activities are modified by the presence of certaindivalent cations; such information is very important when preparingculture media in which to grow the microorganisms and to perform thetest reactions. For example, these yeast-derived phosphatases arestrongly activated by magnesium (Mg²⁺) ions, and are inhibited by zinc(Zn²⁺) ions.

The novel phosphatase enzyme of the present invention was discovered incultures of A. niger. A 4-nitrophenylphosphatase activity wasunexpectedly found in extracts of homogenized filamentous structuresknown as "mycelia." The 4-nitrophenylphosphatase extracted from these A.niger mycelia exhibited optimal enzymatic activity in aneutral-to-slightly alkaline pH environment (i.e., about pH 7.0 to aboutpH 8.5). Similar to the S. cerevisiae enzyme noted above, the A. nigerphosphatase activity is markedly stimulated by magnesium ions, and isinhibited by cations of zinc. While being highly specific for 4-NPP as asynthetic substrate, the neutral A. niger phosphatase has physical andfunctional characteristics which distinguish it from the otherphosphatase enzymes from calf, E. coli, and A. niger. For example, thenew A. niger enzyme exhibits a unique and remarkable substratespecificity; it will interact with the 5'-terminal phosphate of a linearand polymeric DNA molecule, but it unexpectedly will not interact withthe terminal phosphate group of a monomeric ATP molecule which maysimultaneously be present as a phosphate donor in the same reactionmixture. This subtrate specificity is both remarkable and unexpected.

In stark contrast to the other phosphatases mentioned above, this new A.niger phosphatase is very heat labile, being completely inactivated byheating to about 50° C. No denaturing agents, such as the detergent SDS,are needed to facilitate inactivation of this enzyme, as is necessarywith the calf-derived alkaline phosphatase. It is very simple,therefore, to destroy the functional activity of this A. nigerphosphatase (as is especially necessary when the dephosphorylated DNAsubstrate is to be subsequently used in a molecular cloning procedure)without risking damage or change to any of the other molecules containedin the reaction mixture.

Furthermore, this A. niger phosphatase functions best in a reactionenvironment which is neutral to slightly alkaline (i.e., between aboutpH 7.0 to 8.5). This is in significant contrast to the other microbialphosphatases tested which require either strongly acid (pH 2.5 to 5.5)or strongly basic conditions (pH 8.5 to 10) for optimal functionalactivity.

The novelty of this new A. niger phosphatase enzyme will become apparentin the following discussion.

SUMMARY OF THE INVENTION

In accordance with the present invention, a purified heat-labilephosphatase enzyme from the filamentous fungus Aspergillus niger isprovided. This enzyme has a native molecular weight of approximately80,000 daltons, and, under denaturing conditions, consists of twoidentical polypeptide subunits, i.e., an alpha-2 dimer, each subunithaving a molecular weight of approximately 37,000 daltons. Indetermining the native molecular charge of this protein, it is found inelectrofocusing studies to have an isoelectric point (a "pI") ofapproximately 4.6. Its enzymatic activity is optimal under neutral toslightly alkaline culture conditions of between about pH 7.0 to about8.5. Production of A. niger phosphatase activity is not repressed bygrowth on media containing inorganic phosphorus nor is it stimulated bygrowth on media containing very limited amounts of inorganic phosphorus.The functional activity of this enzyme is stimulated in the presence ofmagnesium cations (Mg²⁺), and to a lesser extent by manganese ions(Mn²⁺), but it is significantly inhibited by zinc (Zn²⁺) and by calcium(Ca²⁺).

This novel phosphatase enzyme, isolated by procedures described indetail hereinafter, is useful in hydrolysis reactions in which terminal5'-phosphates are removed from DNA molecules. This hydrolytic removal of5'-terminal phosphate groups from DNA is an enzymatic process called"dephosphorylation." Exemplary of the use of such a dephosphorylationprocess is the removal of 5'-terminal phosphate groups from DNA duringradioactive end-labeling procedures. Another example is thedephosphorylation of linear DNA molecules in molecular cloning assays toprevent self-ligation, i.e., to prevent the spontaneous orenzymecatalyzed ligation of the highly-reactive 5'-terminal phosphate onone end of a linear DNA molecule to the 3'-hydroxyl reactive moiety onthe opposite end of the same molecule (or, alternatively, to the3'-hydroxyl ends of other "bystander" DNA molecules in the mixture.) Yetanother example is the dephosphorylation of RNA molecules forend-labeling in molecular biological studies.

Like other phosphatase enzymes, the functional hydrolytic activity ofthis A. niger enzyme has usually been measured against thenon-biological (synthetic) substrate 4-nitrophenylphosphate (4-NPP),which absorbs blue light when it is hydrolysed. This chemicaltransformation of 4-NPP is easily detected, and is quantifiable in astandard spectrophotometer. Thus, the A. niger phosphatase of thepresent invention exhibits high specificity for 4-NPP, and,correspondingly, for the terminal 5'-phosphate on DNA, which is apolymeric nucleotide molecule. However, the A. niger phosphataseexhibits, remarkably, virtually no reactivity with other substrates,including the monomeric nucleotide ATP. A variety of sugar phosphates,nucleotides, organic phosphates and inorganic pyrophosphates are nothydrolyzed by the A. niger enzyme. Neither does this enzyme possessprotein phosphatase activity.

The removal of 5'-terminal phosphate groups is effected duringprocedures designed to replace the 5'-terminal phosphate group with alabeled phosphate group. In a preferred embodiment, these proceduresinvolve a rapid dephosphorylation and rephosphorylation step, at aneffective temperature and pH, in the presence of a polynucleotide kinaseand an adenosine triphosphate (ATP) molecule bearing a labeled phosphategroup. The labeled phosphate group of some large molecules have beenlabeled with a member selected from the group consisting of aradioactive isotope, an enzyme, a chromophore and a fluorochrome, anddetection of this labeled phosphate group has been by meansappropriately selected from the group consisting of a radiometric means,an enzymatic means, a chromophorometric means and a fluorometric means.However, in a preferred embodiment of the present invention, the labeledphosphate group on ATP is labeled with radioactive phosphorus-32 (i.e.,³² P). This label, and procedures for its use, is discussed in therecent volume by Maniatis T, Fritsch EF, and Sambrook J: MolecularCloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory,1982.

Thus, this new enzyme is distinguished from other phosphatases becauseit is: (1) reactive with DNA, but not with ATP; and (2) heat labile at50° C., even in the absence of denaturing agents such as SDS. At 50° C.,it has a half-life of only 5.3 minutes. These characteristics providethis new phosphatase enzyme with great versatility, compared with otheravailable phosphatase enzymes, for the following reasons:

a. because it does not interact with ATP, this A. niger phosphatase canbe used in molecular end-labeling and/or molecular cloning procedures inwhich phosphate-bearing substrates in addition to DNA are present in thesame reaction mixture; and

b. should it be necessary, the activity of the enzyme of the presentinvention can be simply and completely abrogated by heating the reactionmixture which contains this enzyme to only 50° C.; no special inhibitingligands or denaturing reagents are required to bind and/or inactivatethe enzyme.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a photograph of the polyarylamide electrophoresis geldescribed in Example 1, showing two columns of protein bands, asdiscussed more fully hereinafter. Column "A" shows the stained bands ofproteins used as molecular weight standards, and column "B" shows thestained band of the novel phosphatase of the present invention.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Purification andproperties of a specific 4-nitrophenylhosphatase isolated fromAspergillus niger

The following example demonstrates how the novel phosphatase of thepresent invention was identified, and subsequently isolated from thefilamentous fungal microorganism, Aspergillus niger. It alsodemonstrates how the unique phosphatase enzyme can be used in molecularcloning assays.

Organism and media

Aspergillus niger, strain NRRL-3, was purchased from the American TypeCulture Collection (ATCC, Rockville, Md.; cell culture No. 9029). It wasgrown in liter batches of liquid medium in 2-liter flasks. To determinewhether the production of this novel phosphatase activity by A. nigerwould be affected by the type of culture medium in which the fungus wasgrown, the following different media were tested: Czapeck's medium(containing sucrose) (Komano T, Plant Cell Physiol. 16: 643-658, 1975);phosphate-limited Czapek's medium; glucose-Czapek's medium (an equalmass of glucose substituted for sucrose); and "corn steep liquor"medium. This latter medium contained, per liter of solution: 70 gglucose, 15.0 g calcium carbonate (CaCO₃), 1.12 g zinc sulfate (ZnSO₄.7H₂ O), 0.6 g ammonium sulfate [(NH₄)₂ SO₄ ], 0.26 g sodium phosphate(NaH₂ PO₄), 5.0 ml "corn steep liquor," and distilled water to a finalvolume of one liter. The pH of the latter medium was adjusted toneutrality (pH 7.0) with 1 M sodium hydroxide (NaOH). Flasks of themedia were inoculated with spores from A. niger previously grown onenriched media (Markwell et al., Appl. Microbiol. Biotechnol. 30:166-169, 1989) and incubated for five days at 25° C. on a rotary shakerat 125 revolutions per minute. The dense filamentous fungal structurescalled "mycelia" were separated from the culture supernatant fluid byfiltration through plastic window screening, followed by washing withdistilled water and squeezing to remove excess liquid. The mycelial masswas then processed for enzyme extraction as outlined below.

Enzyme purification

The enzyme of the present invention is readily detected, as describedabove, by its specific hydrolytic dephosphorylation reactivity with thsmall synthetic substrate called 4-nitrophenylphosphate (4- NPP).

Fungal mycelia of A. niger grown in seven liters of corn steep liquormedium were harvested as outlined above. The mycelia (233 g wet weight)were then placed in a 1 liter square bottle with 500 ml of 10 mMTris-hydrochloride (HCl) buffer (pH 8.0), 1 mM magnesium chloride(MgCl₂), at 4° C., and homogenized for 30 seconds with a Polytronhomogenizer (2.0 cm diameter probe) set at its highest speed. After thismixture was centrifuged at 16,000× x g for 15 minutes, the supernatant,containing almost no phosphatase activity, was discarded. The pellet wasresuspended in 400 ml of 10 mM Tris-HCl (pH 8.0), 1 mM MgCl,, 2.5 Msodium chloride (NaCl) at 4° C. and again homogenized for 30 seconds.The homogenate was then filtered through Whatman No. 1 filter paper withvacuum. The filtered crude enzyme solution was stored at 4° C. in lightimpermeable containers until used for final purification of the new DNAphosphatase.

In the initial step of purification of the DNA phosphatase, 200 ml ofcrude enzyme were mixed with 2 ml of 100 mM PMSF (dissolved inisopropanol), and 0.2 ml of 1 M MgCl₂. This was gently shaken, and thecentrifuged for 20 minutes at 17,000 RPM in a Sorval SS34 rotor. Thepellet was discarded, and the clear supernatant fluid was collected andapplied to a 12×2.5 cm column of Phenyl Sepharose CL-4B (Pharmacia)equilibrated in 1O mM Tris-HCl (pH 8.0), 1 mM MgCl₂, 2.5 M NaCl. Thecolumn was then washed at a flow rate of 3 ml min⁻¹ with a step gradientconsisting of the following: (i) 100 ml of 10 mM Tris-HCl (pH 8.0), 1 mMMgCl₂, 2.5 M NaCl; (ii) 100 ml of 10 mM Tris-HCl (pH 8.0), 1 mM MgCl₂,0.2 M NaCl; and (iii) 100 ml of 10 mM Tris-HCl (pH 8.0), 1 mM MgCl₂.Fractions of ten ml were collected. The phosphatase activity, asdetermined in sensitive 4-NPP assays, came off the column as a peak withthe last buffer solution and was pooled based on specific activity.

The pooled phosphatase activity eluted from the

Phenyl Sepharose column was then applied to a 8×2.5 cm column ofQ-Sepharose (Pharmacia) equilibrated with 10 mM Tris-HCl (pH 8.0), 1 mMMgCl,. The column was washed until absorbance at 280 nm returned to zeroas monitored with an Isco V⁴ absorbance monitor. A 400 ml lineargradient of 0.0 to 0.4 M NaCl in 10 mM Tris-HCl (pH 8.0), 1 mM MgCl₂ wasused to elute the phosphatase, at a flow rate of 1 ml per minute.Fractions of seven ml were collected, and the flow rate was 1 ml min⁻¹.The enzyme activity eluted from the column at approximately 0.15M NaCl;fraction numbers 38 to 44 were pooled based on specific phosphataseactivity, as determined in 4-NPP assays.

The pooled fractions from the Q-Sepharose column were concentrated bypressure filtration through a YM-30 Diaflo membrane (Amicon). Thisconcentrated sample was then applied to a 50×2.5 cm column ofSuperose-12 (Pharmacia) equilibrated with 10 mM Tris-HCl (pH 8.0), 1 mMMgCl,, 0.1 M NaCl. Fractions of 2 ml were collected at a flow rate of0.2 ml per minute. Fraction numbers 30 to 36 were pooled and used forfurther experiments. Native molecular size calibration for theSuperose-12 column utilized the following markers: lactate dehydrogenase(M_(r) 135,000), bovine serum albumin (M_(r) 66,000), carbonic anhydrase(M_(r) 29,000) and horse heart cytochrome c (M_(r) 12,400).

Electrophoresis

A 10% polyacrylamide gel containing the powerful, negatively chargeddetergent sodium dodecyl sulfate (SDS) was used to determine purity andmolecular weight of the purified phosphatase. Molecular size standardsused included the following: alpha-lactalbumin (14,200); soybean trypsininhibitor (20,100); trypsinogen (24,000); bovine carbonic anhydrase(29,000); rabbit muscle glyceraldehyde-3-phosphate dehydrogenase(36,000); egg albumin (45,000); and bovine serum albumin (66,000). A 7%acrylamide native isoelectric focusing gel was used to determine theisoelectric point (referred to as "pI"). Standard proteins of knownisoelectric point included the following: amyloglucosidase from A. niger(pI 3.6); glucose oxidase from A. niger (pI 4.2); soybean trypsininhibitor (pI 4.6); bovine milk beta-lactoglobin A (pI 5.1); bovinecarbonic anhydrase II (pI 5.4 and 5.9); and human carbonic anhydrase (pI6.6). The cathode solution was 0.1M beta-alanine and the anode solutionwas 0.1M acetic acid.

Assays

A. niger phosphatase activity was routinely determined by monitoring, ina spectrophotometer set at an absorbance wavelength of 400 nm, theformation of p-nitrophenol generated by the hydrolysis of 4-NPP(purchased from Calbiochem). Non-enzymatic hydrolysis controls wereroutinely included. The phosphatase assay contained the following in atotal volume of 1.0 ml: 8 mM 4-NPP; 25 mM Tris-HCl (pH 8.0); and 5 mMMgCl₂. The reaction, which was initiated by addition of the crude,mycelium-free supernatant fluid mentioned above, was carried out at 30°C. in a Varian DMS 70 spectrophotometer with a wavelength bandpass of 2nanometers. Molar absorptivity of 4-nitrophenol at pH 8.0 was 1.62×10⁴M⁻¹ cm⁻¹.

Assays carried out at pH values other than pH 8.0 were done as end pointdeterminations. For determination of pH optimum, buffer systems usedwere: 25 mM sodium acetate for pH 4.0 to pH 5.5; 25 mM2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol for pH6.0 to pH 6.5; and 25 mM Tris-HCl for pH 7.0 to pH 9.0. The reactionswere terminated by adding 2.0 ml of 100 mM NaOH to the reactionmixtures. The amount of chromogenic 4-nitrophenol produced wasdetermined from the absorbance at a wavelength of 400 nanometers using amolar absorptivity of 1.86×10⁴ M⁻¹ cm⁻¹.

Determination of substrate specificity utilized the assay of inorganicphosphate (P_(i)) by reaction of phosphomolybdate with malachite green(Carter and Karl, J. Biochem. Biophys. Methods 7: 7-13, 1982) or theextraction of released ³² P_(i) into an organic phase (Shacter E, Anal.Biochem. 138: 416-420, 1984). Appropriate controls lacking enzyme samplewere included. Assays were normally carried out in triplicate andincluded samples incubated with calf intestine alkaline phosphatase aspositive controls. For all assays, one unit of activity was defined asthe release of 1 umole phosphate per minute at 30° C. Specific activitywas defined as units per milligram of protein. Protein concentration wasdetermined by the bicinchoninic acid method of Smith et al. (Anal.Biochem. 150: 76-85, 1985).

Exonuclease and endonuclease activities of phosphatase preparations wereinvestigated by incubation of 0.1 unit of A. niger phosphatase witheither 1 microgram of supercoiled pUC8 (BRL) or 1.2 micrograms of HindIII digested phage-lambda DNA (Stratagene). Mobilities of the DNAsamples on 0.8% agarose gel electrophoresis were compared to untreatedsamples. Incubations were carried out in 10 mM Tris-HCl (pH 8.0), 1 mMMgCl₂ at 37° C. for one hour.

Substrate preparation

Histones (Histone III-S) and Phosphorylase-b enzyme were labeled with[gamma-³² P]ATP (ICN Radiochemicals) and the catalytic subunit of thebovine heart cAMP-dependent protein kinase. [Gamma-³² P]ATP was used asa substrate at a specific activity of 0.25 uCi per pmol.

Circular plasmid DNA (pUC8) was opened up and linearized with the use ofthe DNA-breaking enzyme Eco RI (Promega). The linear DNA molecule wasthen dephosphorylated with calf intestine alkaline phosphatase (Promega)and purified by phenol:chloroform:isoamyl alcohol (25:24:1) extractionand ethanol precipitation. The linearized DNA was then 5'-end labeledwith [gamma-³² P]-ATP and polynucleotide kinase (Stratagene).

Unless otherwise noted, all materials were purchased from Sigma ChemicalCo., St. Louis, Mo.

RESULTS Effect of medium on phosphatase production

The effect of different kinds of growth medium on the 4-NPP phosphataseactivity of A. niger mycelia extracts was explored. Five days afterinoculation, cultures were harvested and homogenized in a manner similarto that described for purification of the phosphatase. The extractsexhibited the following amounts of 4-NPP phosphatase activity per 10 gfresh mycelia: Czapek's medium (0.21 unit); phosphate-limiting Czapek'smedium (0.06 unit); glucose-Czapek's medium (0.10 unit); and corn steepliquor medium (1.11 units). Corn steep liquor medium was found topromote the highest production levels of 4-NPP phosphatase activity. Theyield of enzyme from cultures grown in other media was significantlyless, with phosphate-limiting conditions producing the least amount ofphosphatase activity. Based on the greater phosphatase activity from A.niger grown in corn steep liquor medium, this medium was adapted for usein all further studies.

Purification

Mycelia (approximately 233 g wet weight, average) were harvested from 7liters of corn steep liquor medium, homogenized, and phosphatase waspurified by sequential chromatographic fractionation on PhenylSepharose, Q-Sepharose and Superose-12. The purification procedure andenzyme enrichment at each step is summarized 10 in TABLE 1.

                  TABLE 1                                                         ______________________________________                                        Purification scheme for A. niger phosphatase                                          Total    Total   Specific        Fold                                 Purification                                                                          Activity Protein Activity                                                                              Recovery                                                                              Purifi-                              Step    (Units)  (mg)    (Units/mg)                                                                            (%)     cation                               ______________________________________                                        Crude ex-                                                                             15.6      309    0.050    100    --                                   tract                                                                         Phenyl  6.88     8.28    0.832   44.1     17                                  Sepharose                                                                     Q-Sepha-                                                                              5.05     0.74    6.82    32.4    136                                  rose                                                                          YM30 con-                                                                             3.37     0.089   37.9    21.6    758                                  centration                                                                    Superose-                                                                             2.02     0.023   87.8    12.9    1760                                 12                                                                            ______________________________________                                    

Size and isoelectric point

Analysis of the final fraction of purified A. niger phosphatase was doneby polyacrylamide gel electrophoresis under denaturing conditions in thepresence of a detergent (see FIG. 1). As shown in FIG. 1, row B,electrophoresis under denaturing conditions revealed a single band ofprotein in the phosphatase preparation, equivalent to a size of 37,000daltons. The sizes (×10⁻³) of marker proteins (as shown in row A) areindicated on the left hand (vertical) axis of the FIGURE. Approximately2 micrograms of purified phosphatase (lane B) had been loaded onto thegel shown. The gel was stained with silver to reveal the protein bands.

In contrast to the above, elution of non-denatured enzyme from aSuperose-12 column corresponded to a native molecular weight ofapproximately 80,000, indicating that the native protein was a dimer oftwo 37,000 dalton subunits.

In determining the native molecular charge of this protein, it is foundin isoelectric focusing studies to have an isoelectric point (a "pI") ofapproximately 4.6. The isoelectric point (pI) was estimated fromisoelectric focusing in polyacrylamide as 4.6.

pH optimum

The pH optimum of the phosphatase for 4-NPP was determined using theend-point assay already described previously. Buffer systems employedwere 25 mM sodium acetate for pH 4.0 to 5.5, 25 mM2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol for pH6.0 to 6.5, and 25 mM Tris-HCl for pH 7.0 to 9.0. The activity wasevident over a broad pH range from pH 5 to 9 and displayed a maximum atpH 8.0.

Kinetics

The K_(m) of the phosphatase for 4-NPP was determined as 0.77 mM fromdouble reciprocal plots of enzyme velocity versus substrateconcentration. The V_(max) of the purified enzyme was 1.9 units per mgprotein and the turnover number was 108 per second. The phosphataseshowed a first-order inactivation with a half-life of 5.3 minutes whenincubated in 10 mM Tris-HCl (pH 8.0) and 1 mM MgCl₂ at 50° C.

Substrate specificity

In addition to 4-NPP, a variety of nucleotides, sugar phosphates, otherorganic phosphates and inorganic pyrophosphate (PP_(i)) were examined aspotential substrates for the purified phosphatase using aspectrophotometric assay for inorganic phosphate (P_(i)) formation.Substrates were incubated with the enzyme for 15 minutes and the amountof P_(i) liberated was measured and expressed relative to the hydrolysisof 4-NPP assayed with the same procedure (TABLE 2). For purposes ofcomparison, identical incubations were prepared in which an equivalentactivity (using 4-NPP a substrate) of calf intestine alkalinephosphatase was included.

                  TABLE 2                                                         ______________________________________                                        Substrate specificity of A. niger 4-NPP and                                   calf intestine phosphatases                                                                    Relative Activity (%)                                        Substrate          A. niger  Calf Intestine                                   ______________________________________                                        8 mM 4-NPP         100       100                                              1 mM 4-NPP          58       103                                              2'-AMP             <1        134                                              3'-AMP             <1         83                                              5'-AMP             ≦1 102                                              dAMP               <1        121                                              ADP                <1         77                                              ATP                ≦1  80                                              dATP               <1         89                                              2'-GMP             <1         99                                              3'-GMP             <1        122                                              5'-GMP             ≦1 140                                              UMP                <1        113                                              PP.sub.i           <1         ND*                                             Glucose-6-Phosphate                                                                              <1        126                                              Glucose-1-Phosphate                                                                              <1        ND                                               6-Phosphogluconic acid                                                                           ≦1 ND                                               4-Methylumbelliferyl Phosphate                                                                   <1         90                                              alpha-napthylphosphate                                                                           ≦1 108                                              ______________________________________                                         *ND, not determined                                                      

As shown in TABLE 2, significant dephosphorylation of any substrateother than 4-NPP by purified A. niger phosphatase could not be detected,whereas all substrates examined were hydrolyzed by the calf intestinalphosphatase. The A. niger phosphatase also failed to hydrolyze4-nitrophenylacetate or bis-4-nitrophenylphosphate, indicating a lack ofesterase and phosphodiesterase activity, respectively (data not shown).The amount of inorganic phosphate released from 4-NPP was stoichiometricwith the formation of 4-nitrophenol, indicating thattransphosphorylation reactions were not occurring to any significantextent.

To further examine one of these potential substrates at a greater levelof sensitivity, the enzyme was incubated with [gamma-³² P]ATP underidentical conditions and assayed for the liberation of ³² P_(i). Noliberation above background was detected and hydrolysis of ATP appearedto occur at a rate less than one/ten-millionth of that observed with4-NPP. Although ATP was able to inhibit the hydrolysis of 4-NPP,analysis revealed that the inhibition was competitive in nature, with aK_(i) of 1.3 mM for ATP. Because the ATP concentration normally used inradioactive end-labeling reactions is near 1 uM (Maniatis T, Fritsch EF,and Sambrook J: Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Laboratory, 1982), the amount of inhibition of thephosphatase by ATP in a combineddephosphorylation/labeled-phosphorylation reaction would be negligible.

The purified A. niger phosphatase has a subunit size approximately equalto that of the catalytic subunits of many eukaryotic protein phosphataseenzymes, some of which hydrolyze 4-NPP, but when the purifiedphosphatase was incubated with either [³² P]histone or [³²P]-phosphorylase-a under the standard assay conditions, the generationof ³² P_(i) from either phosphoprotein was not observed.

End-labeled linear pUC8 DNA (20 pmol 5'-ends) was incubated with 0.05units of A. niger phosphatase (measured using 4-NPP as substrate) for 1hour under standard assay conditions and 0.27 pmol ³² P_(i) wasliberated. Calf intestine alkaline phosphatase was also incubated underidentical conditions and 12.4 pmol ³² P_(i) was liberated. Controlslacking enzyme were incubated under identical conditions. Hydrolysis ofDNA 5'-phosphate under these conditions occurred reproducibly at a rateof 10⁻² % and 0.4% of that observed with 4-NPP with A. nigerphosphatase, and calf intestine alkaline phosphatase, respectively. Theactivity observed probably represented actual phosphatase activity sincethe purified A. niger phosphatase contained no detectable endo- orexonuclease activity, and the assay, utilizing the formation of aphosphomolybdate complex and its extraction into toluene, wasinsensitive to nuclease activity.

Effect of inhibitors and divalent cations

Prospective inhibitors were added to the standard assay with 4-NPP assubstrate and inhibition was monitored and expressed as percentinhibition of the untreated sample (TABLE 3).

                  TABLE 3                                                         ______________________________________                                        Effect of inhibitors on A. niger phosphatase activity                         Sample            Inhibition (%)                                              ______________________________________                                        untreated          0                                                          1 mM EDTA          8                                                          10 mM EDTA        94                                                          1 mM orthophosphate                                                                             11                                                          5 mM orthophosphate                                                                             26                                                          10 mM orthophosphate                                                                            39                                                          1 mM PP.sub.i      4                                                          10 mM PP.sub.i    56                                                          1 mM sodium fluoride                                                                            22                                                          10 mM sodium fluoride                                                                           80                                                          1 mM iodoacetate  96                                                          1 mM N-ethylmaleimide                                                                           75                                                          ______________________________________                                    

The potent chelator of divalent cations, ethylenediaminetetraacetate(EDTA), was found to be a strong inhibitor of the new phosphatase at 10mM, suggesting a divalent metal requirement for catalytic activity.Orthophosphate and inorganic pyrophosphate produced a limited inhibitionat the concentrations tested whereas sodium fluoride gave stronginhibition. Iodoacetate and N-ethylmaleimide, covalent modifiers ofreactive sulfhydryls, were incubated with the phosphatase for 15 minutesat room temperature prior to assays. Treatments with both iodoacetateand N-ethylmaleimide strongly inhibited phosphatase activity, suggestingthat modification of a sulfhydryl group may negatively affect enzymaticactivity.

After treatment with EDTA, this chelator was removed from the enzyme bygel filtration and the specific activity of the 4-NPP phosphatase wasmeasured following addition of various divalent cations (TABLE 4).Addition of 1 mM Mg²⁺ provided a 20-fold stimulation of activity, butwas only able to restore 39% of the specific activity measured prior toEDTA treatment. Added Mn²⁺ produced a slight stimulation whereas Zn²⁺and Ca²⁺ inhibited the enzymes activity even further.

                  TABLE 4                                                         ______________________________________                                        Effect of divalent cations on A. niger phosphatase                            specific activity                                                             Sample         % of untreated                                                 ______________________________________                                        untreated      100                                                            EDTA treated   2                                                              1 mM MgCl.sub.2                                                                              39                                                             5 mM MgCl.sub.2                                                                              36                                                             10 mM MgCl.sub.2                                                                             36                                                             0.1 mM MnCl.sub.2                                                                            8                                                              1 mM MnCl.sub.2                                                                              8                                                              0.01 mM ZnCl.sub.2                                                                           2                                                              0.1 mM ZnCl.sub.2                                                                            1                                                              1 mM ZnCl.sub.2                                                                              0                                                              0.1 mM CaCl.sub.2                                                                            0                                                              1 mM CaCl.sub.2                                                                              0                                                              ______________________________________                                    

End-Labeling of linear DNA molecules

In the usual end-labeling process, the highly-reactive 5'-terminalphosphate group of the linear DNA molecule is first hydrolyticallyremoved by the dephosphorylating enzyme, after which the activity of thephosphatase is destroyed by heat denaturation, with or withoutadditional treatment with chemical denaturing agents, such as adetergent like SDS. The dephosphorylated DNA is then precipitated,washed, and reisolated free of the phosphatase enzyme and denaturingagents, in preparation for the next series of reaction steps ofrephosphorylation, most commonly with a phosphate group bearing adetectable label. In a preferred embodiment, the labeled phosphate groupis derived from the terminal high-energy phosphate group of aradiolabeled adenosine triphosphate (ATP) molecule. The labeledphosphate group is covalently linked at the 5'-terminal position of thetreated DNA by the action of a polynucleotide kinase.

This dephosphorylation-rephosphorylation process essential to most DNAend-labeling studies, is greatly simplified when the dephosphorylatingenzyme will only react with one substrate, and can, therefore, remain inthe reaction mixture without interfering in the subsequent steps of thereaction sequence. Because of its substrate specificity, such is thedistinctiveness of the novel A. niger phosphatase enzyme of the presentinvention.

Therefore, the great distinction of this process, compared to otherdephosphorylation/labeled-rephosphorylation procedures, is itssimplicity: i.e., the A. niger phosphatase enzyme is not denatured orremoved from the reaction mixture after it dephosphorylates the DNAmolecule, as other phosphatase enzymes must be. Because it will notinteract with the phosphate groups of the ATP, which are utilized in thenext step of the reaction, the A. niger phosphatase is left in thereaction mixture, thereby eliminating at least one, if not two,time-consuming procedural steps in the entire end-labeling procedure.

While the present invention has been described in conjunction withpreferred embodiments and specific examples, the description is notmeant to limit it. One of ordinary skill, with the aid of the presentdisclosure, may be able to effect various changes, substitutions ofequivalents and other alterations to the methods and compositions hereinset forth. Therefore, the protection granted by Letters Patent shouldnot be limited except by the language of the claims as set forth below.

What is claimed is:
 1. A purified, heat-labile, phosphatase fromAspergillus niger, said phosphatase having a molecular weight of about80,000 daltons, an isolectric point of about pI 4.6, and a half-life ofabout 5.3 minutes at about 50° C., exhibiting optimal enzymatic activityunder neutral to slightly alkaline conditions of between about pH 7.0 topH 8.5, removing the 5'-terminal phosphate group from a linear DNApolymeric molecule, and not the phosphate group from a monomericadenosine triphosphate (ATP) molecule, and consisting of two identicalpholypeptides subunits, said subunits having a molecular weight of about37,000 daltons each.